Method for inducing differentiation of human pluripotent stem cell into intermediate mesoderm cell

Abstract

The present invention relates to: a method for producing an intermediate mesoderm cell from a human pluripotent stem cell, comprising a step of culturing the human pluripotent stem cell in a medium containing Activin A and Wnt or a functional equivalent of Wnt and a step of culturing cells in a medium containing BMP and Wnt or a functional equivalent of Wnt; to a method for producing a metanephric cell from the intermediate mesoderm cell produced by the first method; to a human pluripotent stem cell having a foreign reporter gene in the chromosome wherein the gene is expressed interlocked with the expression of endogenous OSR1; to a method for screening for an inducer for differentiation into intermediate mesoderm using the human pluripotent stem cell; and to a kit for inducing the differentiation into an intermediate mesoderm cell.

The invention claimed is: 
     
       1. A method for producing an intermediate mesoderm cell from a human pluripotent stem cell, comprising the following steps (i) and (ii) of:
 (i) culturing the human pluripotent stem cell in a medium containing Activin A and Wnt or a functional equivalent of Wnt, and then 
 (ii) culturing the cell obtained in the step (i) in a medium containing BMP and Wnt or a functional equivalent of Wnt, and 
 (iii) obtaining intermediate mesoderm cells, 
 wherein the culture in the step (i) is performed for a culture period ranging from 2 to 5 days, and 
 wherein the intermediate mesoderm cell is an OSR1-positive cell and is identified by detection of one or more markers selected from the group consisting of: PAX2, WT1, EYA1, and SIX2. 
 
     
     
       2. The method of  claim 1 , wherein in the culture of the step (i), the human pluripotent stem cell is cultured in suspension to form a cell population or cell mass of the human pluripotent stem cell, and in the culture of the step (ii), the cell population or the cell mass is subjected to adhesion culture, thereby forming the intermediate mesoderm cell. 
     
     
       3. The method of  claim 1 , further comprising, in the culture of the step (i), substantially separating the cell population or cell mass of human pluripotent stem cell into respective cells. 
     
     
       4. The method of  claim 3 , wherein step (i) further comprises adhering the separated cells to a Matrigel™-coated dish or a collagen-coated dish and then culturing the cells. 
     
     
       5. The method of  claim 1 , wherein in the step (i), the medium further contains fetal bovine serum (FBS). 
     
     
       6. The method of  claim 1 , wherein in the step (ii), the medium further contains a knockout serum replacement (KSR). 
     
     
       7. The method of  claim 1 , wherein the Wnt is Wnt3a. 
     
     
       8. The method of  claim 1 , wherein the functional equivalent of Wnt is a GSK3β inhibitor. 
     
     
       9. The method of  claim 8 , wherein the GSK3β inhibitor is CHIR99021. 
     
     
       10. The method of  claim 1 , wherein the BMP is BMP7. 
     
     
       11. The method of  claim 2 , wherein the culture in the step (ii) is performed for a culture period ranging from 14 to 18 days. 
     
     
       12. The method of  claim 3 , wherein the culture in the step (ii) is performed for a culture period ranging from 7 to 10 days. 
     
     
       13. The method of  claim 1 , wherein the human pluripotent stem cell is a human iPS cell or a human ES cell. 
     
     
       14. The method of  claim 1 , wherein the human pluripotent stem cell has a foreign reporter gene in the chromosome, wherein expression of the gene is interlocked with the expression of endogenous OSR1. 
     
     
       15. The method of  claim 14 , wherein the reporter gene is a DNA encoding a fluorescent protein, a luminescent protein, GUS, or LacZ. 
     
     
       16. The method of  claim 14 , wherein the pluripotent stem cell is a human iPS cell.